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November 1952 E-849 

i^rr nfiiftij&d States Department of Agriculture 
STATE "Lr" Agricultural Research Administration 

Bureau of Entomology and Plant Quarantine 



STUDIES OF THE STABILITY OF TOXAPHENE EMULSIONS 

IN DIPPING VATS!' 

ByB.I. Sparr, J.C. Clark, E.E. Vallier, andA.H. Baumhoverl/ 
Division of Insects Affecting Man and Animals 



Several reports have been received of cattle killed by dipping in 
emulsions that contained apparently safe concentrations of toxaphene 
when tested by the vat-side method (Bowen 1). In the belief that the 
stricken animals had taken from the vat more than a safe quantity of 
toxaphene, a study was made of the stability of these emulsions in the 
vats. A threefold program was set up. The first phase consisted of 
actual dipping on the ranch. Another aspect was a photomicrographic 
study of the dip contents. The third phase consisted of an analysis of 
hair samples from dipped cattle to determine the quantity of toxicant that 
adhered to the hair. R. D. Radeleff (unpublished work) had shown that 
more toxaphene tended to be deposited on animal hair from a coarse 
emulsion than from a fine emulsion. He used oil-soluble, radioactive 
1, l,l-trichloro-2,2-bis(4-chloro-3-iodophenyl)ethane as an indicator, 
dissolving a small quantity in the emulsifiable concentrate to be tested. 

Three experimental concentrates were chosen for this study because 
they produced stable emulsions in laboratory glassware- -the Kerrville 
concentrate, formulated by the authors, and two concentrates, SP-159 
and SP-141, supplied by Wm. Cooper & Nephews. The percentage com- 
position of each is as follows: 

Concentrate Toxaphene Kerosene Emulsifier 

Kerrville 50 40 Duponol G, 10 

SP-159 62 25.5 CTX-54, 12.5 

SP-141 61 24 CTX-141,15 



1/ Report of a study made under the Research and Marketing Act 
of 1946. 

2/ The authors are indebted to R.C. Bushland and H. V. Claborn,of 
this Bureau, and to R. D. Radeleff, of the Bureau of Animal Industry, 
for help and advice. 



- 2 - 

Procedures 

Dipping . --Three vats were charged, one with each concentrate pre- 
mixed with a little water before dilution to a final concentration of ap- 
proximately 0.5 percent of toxicant. This required a 100-to-l dilution 
of the Kerrville concentrate and 150-to-l dilutions of SP-159 and SP-141. 
Animals were dipped at various intervals, and hair samples were taken 
before and after dipping. Samples of the emulsion were taken periodically 
for vat-side tests and microscopic study. When necessary, additional 
water or toxicant was added to the vats to maintain a suitable volume 
and the 0.5 -percent toxaphene concentration. 

Photomicrography . --The equipment consisted of a monocular re- 
search microscope, equipped with an ocular grid and micrometer and a 
1.9 -mm. oil-immersion objective; a bellows-type photomicrographic 
camera; Kodak film --Super Panchro Press, type B; a microscope lamp 
with a 100-watt projection bulb; microscope slides and cover slips. 

A grease ring was drawn on the slide and sufficient sample added 
barely to fill the circle. A cover slip was mounted over the grease ring 
to form a seal. The slide was then placed on the microscope, the micro- 
meter of which had been calibrated. (At a magnification of 950 diameters, 
one scale division equals 1.5 microns.) The field to be photographed was 
chosen so as to be a representative view with as little extraneous mate- 
rial as possible. This view was then photographed, usually at 1/50 
second, and the film was immediately developed. 

Analysis of hair for toxaphene retention . --Three-gram samples of 
hair clipped from the sides of Hereford cattle were placed in 100-ml. 
wide-mouthed Florence flasks. To each flask 75 ml. of analytical- grade 
benzene was added. The flask was stoppered, gently swirled, and 
allowed to stand for 24 hours. Each benzene solution was filtered through 
fat-extracted cotton to obtain a 45 -ml. aliquot and then subjected to 
routine analysis for organic chlorine. On the basis of the number of 
equivalents of chlorine present in each sample, the percentage of toxa- 
phene was calculated. 

Kerrville Concentrate 

Dipping. --A 2,700-gallon vat was charged on May 8, 1951, and used 
for dipping cattle and newly shorn sheep and goats. Dippings were as 
follows: 

May 8 98 cattle 

15 461 goats 

June 18 87 cattle 

23 535 sheep 

25 319 sheep 

27 ----- - 497 goats 

July 12 10 cattle 



-3- 

This dip obviously deteriorated rapidly. At the time of the second 
dipping the emulsion had creamed to the bottom, but it was readily 
redispersed by dragging a bucket through the vat. On June 18, when 
the dip was approximately 6 weeks old, it showed evidence of further 
deterioration, as traces of free oil appeared when the contents were 
agitated. On July 12 considerable free oil came to the surface as 
cattle passed through the vat, and dipping was discontinued after 10 
cattle had been dipped, since the dip was considered unsafe for further 
use. 

Photomicrographic study . --Figure 1 shows the freshly prepared 
emulsion. A few of the particles are more than 1.5 microns in diameter, 
but most of them are smaller. Owing to difficulties in technique, the 
camera is not able to capture a large number of the smaller particles 
that are discerned when this emulsion is viewed through the microscope, 
at which time the light intensity is considerably less. 

Figure 2 shows the emulsion when it is deteriorated. This sample 
was taken from the vat on July 12. Here there are many particles more 
than 1.5 microns in diameter. A tendency for the particles to aggregate 
is shown in the large numbers of clusters of varying size. This is a 
fairly true reproduction of the appearance of the emulsion when viewed 
through the microscope and the illumination is decreased. Those small 
particles that are seen through the microscope but not photographed in the 
fresh emulsion are not present in the deteriorated emulsion. 

Analysis of hair for toxaphene retention . - -1 he percentages of toxaphene 
retained by the hair of the cattle are shown in table 1. 

Concentrate SP-159 

Dipping. --A 2, 800 -gallon vat was charged with concentrate SP-159 
on May 10, 1951, and used for dipping cattle and newly shorn sheep and 
goats. Dippings were as follows: 

May 10 89 cattle 

27 143 sheep 

June 5------ 73 cattle and 104 goats 

July 3 76 cattle 

August 1 - - - - - 78 cattle 

September 11 79 cattle 

October 25 - - - - 54 cattle 

November 8- - - - 11 cattle 

Until August 28 the vat contents appeared unchanged. On September 11 
the emulsion had a greasy appearance, but no free oil was seen. The volume 
and strength of the dip were maintained by adding water and toxicant when 
necessary. 



On September 27 free oil was brought to the surface for the first 
time by dragging with the bucket. A sample of dip taken from the sur- 
face of the vat before stirring tested 0.17 percent of toxaphene. Here- 
tofore similar samples had shown more than 0.30 percent. There had 
been no appreciable rainfall. On October 25 considerable free oil was 
thrown to the surface and persisted throughout the dipping. On 
November 8 the appearance of the dip had not changed significantly. 

Photomicrographic study . --Figure 3 depicts the freshly prepared 
emulsion. Again many small particles that do not appear on this photo- 
graph could be viewed under a dim light. All the particles in this emul- 
sion undergo rather active Brownian movement. Figures 4, 5, and 6 
depict the emulsion on August 28, September 11, and October 25, 
respectively. The differences in size distribution are distinct. Both 
figures 5 and 6 show several particles more than 3 microns in diameter, 
which appear to be packed with smaller particles. 

Analysis of hair for toxaphene retention . --The percentages of toxa- 
phene retained by the hair of the cattle are shown in table 1. 

It should be noted that the toxaphene retention in this deteriorating 
emulsion is not quite so high as in the emulsion prepared from the 
Kerrville concentrate. On November 8 the SP-159 dip was in better 
condition than the Kerrville concentrate at the time it was discarded. 

Concentrate SP-141 

Dipping. --A 1,900 gallon vat was charged with concentrate SP-141 
on May 22, 1951, and used for dipping cattle only. The numbers dipped 
were as follows: 

May 22 - - 100 

June 7 - - 104 

25 104 

July 10 106 

25 106 

August 15 93 

September 12 100 

October 16 46 

November 8 - - - 10 

There were no changes in the gross appearance of the dip until 
September 12, when it failed to foam during dipping. During previous 
dippings there had been considerable foaming. Free oil was first noted 
on September 28, when the dip was 18 weeks old. This oil redispersed 
upon agitation. On October 16 the oil initially brought to the surface 
appeared to re-emulsify with continued dipping. On October 24 there 



were larger quantities of free oil, which would not re-emulsify with 
manual agitation. A sample from the top of the undisturbed dip contained 
0.24 percent of toxaphene, whereas earlier samples similarly taken con- 
tained more than 0.30 percent. On November 8, at the last dipping, the 
appearance of the dip had not changed. 

Photomicrographic study . --The freshly prepared emulsion showed 
very few particles. Figure 7 is a fairly true reproduction of what is 
actually visible. A comparison with figures 1 and 3 shows this dip to 
be the finest emulsion of the three under discussion. Observations of 
creaming rates in the laboratory support this conclusion. 

Figure 8 represents the dip on September 7. Note the change in 
particle- size distribution. Figure 9 depicts the dip after the sixth dipping, 
on September 12; again the change in size distribution is marked. As in 
figures 5 and 6, a number of the large particles are filled with smaller 
particles. 

Analysis of hair for toxaphene retention. --The percentages of toxa- 
phene retained by the hair of the cattle are shown in table 1. 

Later Dippings in Mixture of SP-159 and SP-141 

Until the October 16 dipping there did not appear to be any significant 
changes. Since the last two dippings in SP-159 and SP-141 were made 
after the cattle had acquired their heavy winter coats, several cattle 
were dipped in a vat freshly charged in November with a mixture of equal 
parts of SP-159 and SP-141 to determine whether there was a difference 
in retention due to the difference in the coat. Hair samples taken from 
two of these animals had 0.66 and 0.77 percent of toxaphene. Two weeks 
later two other animals were dipped and their hair tested 0.89 and 0.85 
percent of toxaphene. Since these analyses do not run higher than the 
hair analyses from the cattle dipped when the vats were freshly charged 
in the spring, it is reasonable to assume that the increase in retention 
in these later dippings is due to change in the nature of the emulsion and 
not in the nature of the animals' hair. 

Conclusions 

Time and use brought about changes in the physical nature of all 
three emulsions. Since deterioration and the consequent increase in 
particle size resulted in heavier deposits of toxicant on the hair of 
dipped animals, they may account for cattle losses following dipping at 
seemingly safe concentrations of toxaphene in dips not completely broken. 

The observation that the amount of insecticide deposited in the hair 
of clipped cattle varies with the particle size of the dispersed phase 
should also be considered in interpreting the results of dipping tests on 
ectoparasite control. Mere maintenance of uniform insecticide 



concentrations in dips may not assure uniform treatment of test animals. 
Therefore, hair analyses should be considered in planning comparisons 
of emulsified insecticides applied as dips. 

Although we have not previously examined the emulsion stability in 
dipping vats so critically as in this experiment, we believe that both 
concentrates SP-159 and SP-141 are superior to formulations with which 
we had previously charged vats. The Kerrville concentrate is unsatis- 
factory. In the tests reported concentrates SP-159 and SP-141 appeared 
to be safe throughout one dipping season, as the toxaphene retention at 
the end of the season was only 50 percent higher than at the beginning, 
and earlier work (Radeleff and Bushland 2) had suggested a 2-to-l margin 
of safety for fresh toxaphene dips. No conclusions can be made regarding 
the general use of either of these concentrates, since they were not tried 
under a variety of conditions. 

Summary 

Observations on three ranch dipping vats, charged with different 
toxaphene emulsifiable concentrates- -a Kerrville concentrate and two 
experimental concentrates, SP-159 and SP-141, supplied by Wm . Cooper & 
Nephews--are described. 

The dips were mixed to contain 0.5 percent of toxaphene and were 
used to treat cattle, sheep, and goats. At each dipping the toxaphene 
concentration was determined by vat- side analysis, and uniform dip 
volume and toxaphene content were maintained by adding appropriate 
amounts of emulsifiable concentrate and water. 

All dips when freshly prepared were such fine emulsions that few 
oil droplets were visible at 950 diameters' magnification. Microscopic 
examinations showed that during a season's use there were visible 
increases in particle size of the dispersed phase. 

Hair clipped from cattle dipped in fresh emulsions contained about 
0.8 percent of toxaphene. As the particle size of the oil phase increased, 
the amount of toxaphene deposited on the coat became greater. With one 
emulsion the toxaphene retention doubled and with the other two emulsions 
it increased one-third in a single dipping season. 

These observations may explain ranch losses of cattle dipped in 
seemingly safe concentrations of toxaphene. They also suggest that hair 
analyses be employed as a check on uniformity of treatment in experi- 
mental comparisons of emulsion dips. 

The dip made with the Kerrville concentrate was considered unsafe 
after 2 months. The two dips made from concentrates SP-159 and 
SP-141 appeared to be satisfactory for an entire season but obviously 
changed in 6 months' use. 



- 7 - 
Literature Cited 



(1) Bowen, C. V. 

195 °- A vT B z:z: t:^:: r ng r aphene in ««• «*-. 

"' • xim. and Plant Quar. ET-285, 9 pp , 
(2) Radeleff, R . D ., and Bushland< R c 



Table 1. --Results of chemical analyses to determine toxaphene 
in hair samples clipped from animals dipped in three experi- 
mental concentrates, each diluted to contain 0.5 percent of 
toxaphene 



Concentrate 


Date of dipp: 


mg 


Age of dip 


Toxaphene 










Weeks 


Percent 


Kerrville 


May 8 






Fresh 


0.68 
.81 




June 18 






6 


1.32 
1.98 




July 12 






9 


1.65 
1.63 


SP-159 


May 10 






Fresh 


0.91 
.81 




August 1 






12 


.71 
.83 




Septemb 


er 


11 


18 


1.25 
1.01 




October 


25 




24 


1.37 
1.21 




November 


8 


26 


1.40 












1.38 


SP-141 


May 22 






Fresh 


1.02 
1.10 




June 7 






2 


1.06 
0.76 




25 






5 


.91 




July 10 






7 


.86 
.99 




25 






9 


1.11 




Septemb 


er 


12 


16 


.94 
.84 




October 


16 




21 


1.23 
1.45 




November 


8 


24 


1.28 












1.39 



-9- 
















a II 


* 









Figure 1. --Fresh emulsion. 

Digitized by the Internet Archive 

in 2013 



Figure 2. --Deteriorated emulsion. 



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